Despite many recent advances in gene therapy methods, effective therapeutic delivery of genes into various cell types, in particular in vivo delivery, has not been achieved simply because methods are not available to cause delivery of therapeutically effective amounts of such genes into the particular cells of a patient in need of treatment. Efficient delivery of therapeutically sufficient amounts of genes, as well as other therapeutic molecules, often has proved difficult, if not impossible, since, for example, the cell membrane presents a selectively-permeable barrier. Additionally, even when genes, or other biologically active molecules, successfully enter targeted cells, they may be degraded, inappropriately transported or, in the case of genes, may fail to be transcribed properly.
One example of a target cell type for which effective delivery methods, in particular for gene therapy, are lacking is the urothelial cells of the bladder. Such methods would be particularly useful in the treatment of bladder cancer. Despite advances in endoscopic and intravesical chemotherapeutic procedures, superficial bladder cancer still has a high recurrence and progression rate (Nseyo U O, Lamm D L. (1996) Semin. Oncol. 5:598-604). Currently, the most successful treatment involves weekly instillation of ‘live’ Bacillus Calmette-Guerin (BCG) into the bladder for two hours (Morales, A. et al. (1976) J. Urol., 116: 180-183; Brosman, S. A. (1992) Urol. Clin. North Am., 19:557-564). Although effective, with routine response rates of 60-70%, side effects such as dysuria, haematuria, frequency and cystitis are common and sometimes severe (Lamm, D. L. (1992) Urol. Clin. North Am., 19:565-572). Moreover, a significant number of patients do not respond to BCG therapy and toxicity is common. Exploration of the mechanism of BCG activation of the immune response has resulted in the identification of cytokines, co-stimulatory molecules and adhesion molecules which play important roles in facilitating the cytotoxic response against tumors (Taniguchi, K et al. (1999) Clin. Exp. Immunol., 115:131-5, 1999; Patard, J. J. et al. (1998) Urol. Res., 26:155-9; Kurisu, K. et al. (1994) Cancer Immunol. Immunother, 39:249-53; Sander, B. et al. (1996) J. Urology, 156:536-41; Chow, N. H. et al. (1998) Urology, 52:1015-9; Jackson, A. M. et al. (1995) Clin. Exp. Immunol., 99:369-75).
Superficial bladder cancers have some features that make them particularly attractive for in vivo gene therapy namely that tumors are often localized and therapeutic genes can be placed in direct contact with the tumor through simple intravesical administration. Further, the response of the tumor to treatment can be easily determined with cystoscopy and urine cytology. One of the major obstacles to successful transfection of the transitional cell epithelia is the presence of a glycosaminoglycan layer, which may act as a significant barrier the uptake of DNA complexes (Ruponen, M. et al. (1999) Biochim. Biophys. Acta, 1415: 331-41).
Viral expression vectors have been used to introduce specific genes locally to the bladder (Sutton, M. A. et al. (1997) Urology, 49: 173-80; Lee, S. S. et al. (1994) Cancer Res., 54:3325-8). However, viral vectors have a number of limitations in a clinical setting such as immunogenicity and safety. A recent study by Li et al. has questioned the effectiveness of adenovirus-based gene therapy for bladder cancer owing to the differences in viral receptor levels observed in human bladder cancer cell lines (Li, Y. et al. (1999) Cancer Res., 59:325-30).
An alternative method of gene therapy involves liposome-mediated delivery of DNA into cells. The advantage of a non-viral system is that it is not receptor dependent and should therefore be applicable to all tumors. Brigham et al. first reported delivery of DNA into tissues using cationic liposomes (Brigham, K. L. et al. (1989) Am. J. Med. Sci., 298:278-81). Since then cationic lipids have been shown to be efficient carriers for localized and systemic delivery of DNA to tissues in vivo (Plautz, G. E. et al. (1993) Proc. Natl. Acad. Sci. USA, 9:4645-9; Nabel, G. J. et al. (1993) Proc. Natl. Acad. Sci. USA, 20:11307-11; Zhu, N. et al. (1993) Science, 261:209-11). Cationic liposomes have a number of important advantages over viral gene delivery systems in the clinical setting. These include the ability to use a range of gene constructs from simple plasmids to chromosomal fragments and fewer safety concerns. Their principal disadvantage, however, is their relatively low transfection efficiency when compared to viral techniques.
Accordingly, methods for increasing transfection efficiencies, particularly of urothelial cells, are still needed, as are general methods for delivering pharmaceutical agents to the bladder.